Use of Ginsenosides in the Treatment of Aids

ABSTRACT

The present invention relates to the use of compounds of formula (I) in the treatment of AIDs, 
     
       
         
         
             
             
         
       
     
     wherein, R 1  is -Glc 2 -Glc, R 2  is selected from the group of -Glc 6 -Glc, -Glc 6 -Ara(p), -Glc 6 -Xyl and -Glc 6 -Ara (f); and R 3  is H, and also relates to the composition comprising the above compounds and the use of the herbal extracts in the treatment of AIDS.

FIELD OF THE INVENTION

The invention relates to a drug for treating acquired immune deficiencysyndrome, AIDs, specifically, the invention relates to the use ofGinsenosides extracted from Ginseng for treating AIDs. The inventionalso relates to the use of Ginsenosides-containing composition for thetreating AIDs. Furthermore, the invention relates to the use ofGinsenosides-containing root, stem and leaf extracts of plants selectedfrom Panax ginseng C. A. Mey, Radix panacis quinquefolii, gymostemmapentaphyllum (thumb) makino, Panax notoginseng (burk) F. H. Chen, Panaxjaponicus C. A. Meyer and Panax japonicus var: major (burk.) Wu et Fengin the treatment of AIDs.

BACKGROUND OF THE INVENTION

With sweet and slightly bitter tastes as well as a mild action, theginseng roots are capable of adjusting the energy flows, nurturing theblood, quieting the spirits, sharpening the wisdom, generating thesaliva, relieving the cough, replenishing the nutrients andstrengthening the body. Known as “the King of Herbs”, it has been thepreferable choice of body-regulating drug ever since the ancient timesin China. It is reported that Ginseng has the following efficiency:

-   1. Ginseng has a sedative effect on central nervous system, and    antagonistic action to a number of analeptic, and reduces inhibitory    effect of central depressive drugs. Rb series compounds of    Ginsenosides have sedative effect on central nervous system, whereas    Rg series compounds have weak excitable effect, but shows inhibitory    effect under over-dose. Ginseng can not only improve the excitable    process of central nervous system, but also strengthen the    inhibitory process, together with centralizing inhibition and    differentiating completely.-   2. Ginseng has anti-fatigue effect. Panaxadiol, panaxtrol and all    kinds of Ginsenosides have effect of anti-fatigue, wherein the    activity of panaxtrol is more than 2 times of that of panaxadiol. It    is believed that ginseng pasty extract liquid can promote economic    use of glycogen and high energy phosphate compound, can enhance the    metabolism of lactic acid and pyruvic acid, and provide energy    timely by oxidation for the muscular movement.-   3. Ginseng increases blood pressure with low dose and lowers blood    pressure with high dose for anesthetic animals. But a therapeutic    dose has no effect on patient's blood pressure; Ginsenosides have    slightly and shortly effect on lowering blood pressure. Ginseng    water extract has the same effect as cardiac glycosides,    strengthening the contraction frequency and slowing heart rate.-   4. Ginseng enhances nonspecific resistance of human body to many    deleterious factors. It can, for example, strengthen the resistance    of human body to invasion from physical (cool, high temperature,    over exercise, high pressure or low pressure), chemical (all kinds    of toxicants, narcotics), biological (foreign serum, microorganism,    transplanting tumor) factors,-   5. Ginseng has blood glucose reduction effect in normal rabbits, as    well as in rats and dogs induced to high blood glucose with alloxan    tetrahydrate, adrenalin, but nevertheless cannot be used as the    substitute of insulin.-   6. Ginseng has the effect like gonadotropin, wherein ginsenosides A,    C, F all have the activity of gonadotropin with similar intensity.-   7. Panax Ginseng PE has no activity of hemolysis, but weak    anti-hemolysis effect that is induced by Ginsenosides or lecithin.    It is reported recently, the elements of Rh, Rg and Rf of    ginsenosides (that is, saponins with panaxtrol being the genin) have    the activity of hemolysis, whereas the elements of Rc and Rb (that    is, saponins with panaxdiol being the genin) have the activity of    anti-hemolysis.-   8. Injection prepared by ginseng extract can increase the content of    erythropoietin in bone marrow of the rabbit. No matter being orally    administrated or externally applied, Ginseng extract can improve the    biologically synthesis of DNA, protein, lipid in the bone marrow    cell, at least parts of the active elements of the Ginseng extract    are Ginsenosides (especially Rb2, Rg1, etc). After being    administrated intra-gastricly, Ginseng extract exhibits the activity    of antidiuresis.-   9. Ginseng has favorable effect on the function of pituitary adrenal    system, showing that it can not only enhance the resistibility of    animals to disadvantage conditions (such as high temperature, low    temperature, long time swimming, etc), but also alleviate the change    of adrenal hypofunction induced by stress reaction.-   10. Ginseng can also improve the synthesis of protein and nucleic    acid. It has been reported recently that Ginseng extract can greatly    improve the synthesis of nucleic acid and protein in liver, kidney,    bone marrow, spermary cells of rats, as well as the synthesis of    serum protein.

As demonstrated by many experiments, ginseng can enhance the physicaland intellectual activities of animals and human and boost the body'snon-specific resistance against a variety of noxious stimulations.Within therapeutic range, it has no interference with the normalphysiological functions and no side effects. It is considered as a classof beneficial and harmless strengtheners and tonics for the whole body.

According to the discovery of the said pharmacological effects ofginseng, the researchers have conducted researches of the ginsengextracts. It is found that more than 10 kinds of Ginsenosides exist withmajor pharmacological actions, such as Rb1, Rb2, Rb3, Rc, Rd, Re, Rg,Rh1, Rh2, F2, pseudo-ginsenoside F1, RTs and American Ginsenoside L1.The main pharmacological studies of these Ginsenosides include theeffects of anti-aging, immunity-boosting and blood lipid-lowering, andsome changes of heart and blood vessels. But until now, there is stillno report of employing ginseng, ginseng extracts or any Ginsenoside totreat AIDS. The causative agent of AIDS is Human Immunodeficiency Virus(HIV), which mainly attacks the human immune system, especially the CD4lymphocyte. At last, the body's immune functions are destroyed,resulting in opportunistic infections and causing the patient's death.

Suramin was the first reported drug to fight against the HIV. In 1985,AZT was found to possess the anti-HIV activity in vitro. In 1986, theclinical trial was carried out. In 1987, AZT became the first drugapproved by FDA for treating AIDS. But the main issues were its drugtoxicities and resistance. Other drugs appeared in the subsequent years.Until now, more than twenty anti-HIV drugs have been approved forcommercial uses in the US. According to their mechanisms of action, theyfall mainly into 3 categories. Except for T20-blocking the entry of HIVinto the cells approved at the end of Year of 2002, all the other drugsbelong to the viral reverse transcriptase (RT) inhibitors, such as AZT,DDC, DDI and viral protease inhibitors. FDA had already approved fivedrugs of protease inhibitors, namely Saquinavir, Ritonavir, Indinavirsulfate and Nefinavir, etc. In 1995, the American scientists adopted the“Triple Combination” regimen of two RT inhibitors and one proteaseinhibitor. Known as HAART, such therapy is currently in common uses.This therapy has improved the treatment outcome and further prolongedthe patient's life. It has been now used for 10 years, several patientsstill survive.

There are currently three drugs available for the cocktail of HAART inChina. But there are only RT inhibiting drugs that have toxicity soserious that nearly 20% of the patients cannot tolerate. Therefore thereexist the issues of drug toxicities and resistance in the clinicalapplications. As stated above, T20 is able to block the entry of virusinto the cells. However, T20 cannot be taken orally since it is apeptide. It must be injected for application. And the price is quiteexpensive. Therefore it is imperative to develop anti-HIV drugs with lowtoxicities and ability of inhibiting drug-resistant HIV.

The traditional Chinese medicine is such a great treasure-house that itis quite worth to be explored and carried forward. Through more than tenyears' researches, the inventors have found out that some herbalextracts, ingredients or mono-components have well-defined anti-HIVactivities. The targets of their anti-HIV activities were studied. Withsuch advantages as a cheaper price and lower toxicities over HAART, theycan markedly boost the immune functions. And it is necessary toconstantly update the therapeutic cocktail since HAART has the problemof drug resistance. So there are broad prospects of applying traditionalChinese medicine to treat AIDS in clinical settings.

SUMMARY OF THE INVENTION

The present inventor has found out that dammarane type compounds (alsois known as tetracyclic triterpene dammarane) among ginseng extracts canbe used for treating AIDS with better efficiency.

Therefore, on one hand, the present invention relates to the use ofcompounds of formula I in the preparation of drugs for treating AIDS:

wherein, R₁ is -Glc²-Glc, R₂ is selected from the group of -Glc⁶-Glc,-Glc⁶-Ara(p), -Glc⁶-Xyl and -Glc⁶-Ara(f); and R₃ is H. Preferably, R₁ is-Glc²-Glc, R₂ is -Glc⁶-Xyl, and R₃ is H.

The prefer dose of present compounds is in amount of 0.03-0.50 mg/kgbody weight.

Present compounds can be administered through oral way, intra-dermalway, injection, inhale or mucosal way.

Present compounds may be used in combination with other anti-HIV drugsin a synergistic way including such as AZT, DDC, DDI, SaquinavirRitonavir, Indinavir sulfate and Nefinavir or combination thereofpreferably AZT. Said compounds are also active against HIV virus that isresistant to some drugs repeatedly used.

On another aspect, present invention relates to the use ofpharmaceutical composition comprising the compounds of formula I asactive ingredient in the preparation of drugs for treating AIDs:

wherein, R₁ is -Glc²-Glc, R₂ is selected from the group of -Glc⁶-Glc,-Glc⁶-Ara(p), -Glc⁶-Xyl and Glc⁶-Ara(f); and R₃ is H.

Furthermore, present invention also relates to the use of root, stem orleaf extracts of Panax ginseng C. A. Mey, Radix panacis quinquefolii,Gymostemma pentaphyllum (thumb) makino, Panax notoginseng (burk) F. H.Chen, Panax japonicus C. A. Meyer and Panax japonicus var: major (burk)Wu et Feng and/or dry powder thereof in the preparation of drugs fortreating AIDs, said extracts comprise the compound of formula I:

wherein R₁ is -Glc²-Glc, R₂ is selected from the group of -Glc⁶-Glc,-Glc⁶-Ara (p), -Glc⁶-Xyl and -Glc⁶-Ara(f); and R₃ is H.

The compounds of formula I disclosed in present invention and/orextracts comprising the compounds of formula I are all active to thevirus resistant to AIDs. Said extracts may be obtained from the root,stem and/or leaf of the plants mentioned above, still further, drypowder made from these plants is also included in the present technicalsolution of the invention. The extracts made from the root, stem or leafof Panax ginseng C. A. Mey, Radix panacis quinquefolii, Gymostemmapentaphyllum (thumb) makino, Panax notoginseng (burk) F H Chen, Panaxjaponicus C. A. Meyer and Panax japonicus var: major (burk) Wu et Fengcan be used in combination with the dry powder of these plants.

Present invention also relates to a pharmaceutical compositioncomprising present compounds or extracts as active element, and ordinarypharmaceutically acceptable excipient or supplementary agents.

Present compounds may be prepared by well-known methods of this field.For this purpose, if necessary, present extracts or compounds may becombined with one or more solid or liquid excipients to prepare intosuitable administration formulation.

Present extracts or compounds or pharmaceutical composition comprisingthereof may be administrated in unit dosage form, and administratingmethod may be intestine or parenteral, such as oral, muscle,subcutaneous, nasal, bucccal mucous member, skin, peritoneum or rectumetc, preferably oral.

The administrating method of present extracts or compounds orpharmaceutical composition comprising thereof may be injection includingmainly intramuscular injection, hypodermic injection and intradermalinjection.

The dosing formulations may be liquids or solids. For example, theliquid formulations can be true solution types, colloid types,microgranular forms, emulsion forms and suspensive forms. Otherformulations include tablets, capsules, drops, aerosols, pills, pellets,solutions, suspensions, emulsions, granules, suppositories andfreeze-dry powder injections and the like.

Present extracts or compounds may be made into common preparations,sustained-release preparations, controlled-release preparations,targeted preparations and varieties of mircosomal drug delivery systems.

All carriers known in the art can be used so as to prepare the unitdosage formulations into the tablets, e.g. carriers as dilutes andabsorbents including starch, dextran, calcium sulfate, lactose,mannitose, sucrose, sodium chloride, glucose, urea, calcium carbonate,white clay, microcrystal cellulose and aluminum silicate, etc; lubricantand adhesive, such as water, glycerol, polyethylene glycol, ethanol,propanol, starch slurry, dextran, syrup, honey, glucose solution,Arabian glue, gelatin glue, carboxylmethylcellulose sodium, sdshellac,methylcellulose, potassium phosphate and PVP, etc; disintegrant, such asdry starch, alginate, agrose powder, algin starch, sodium bicarbonate,citrate, calcium carbonate, polyoxyethylene sorbitan alphate, sodiumdodecylsulfonate, methylcellulose and ethylcellulose, etc;disintegration inhibitor, such as sucrose, glycol tristearate, cocoabutter and hydrogenated oil, etc; absorption promoter, such asquaternary ammonium and sodium dodecylsulfate, etc; lubricant, such astalcum powder, silicon oxide, corn starch, stearate, borate, liquidparaffin and polyethylene glycol, etc. Other carriers, such aspolyacrylic acid resins and liposome; water-soluble carriers, such asPEG4000, PEG6000 and PVP, etc can also be used. Also the tablets may befurther prepared into coated pills, such as sugarcoated, thinmembrane-coated, enteric coated, or double-layered and multi-layeredtablets.

For example, it is feasible to adopt extensively all the known carriersin the art so as to prepare the unit dosage formulations into thepellets. Examples of carriers are dilutes and absorbents, such asglucose, lactose, starch, cocoa butter, hydrogenated plant oil, PVP,kaolin and talcum powder, etc; adhesive, such as Arabian glue,tragacanth gum, gelatin, ethanol, honey, liquid sugar and rice or flourpaste, etc.; disintegrant, such as agrose powder, dry starch, alginate,sodium dodecylsulfonate, methylcellulose and ethylcellulose, etc.

For example, with the purpose of preparing the unit medicine into thecapsules, the active ingredients of the invented medicinal compositioncan be mixed with various said carriers to obtain a mixture. And themixture is placed into hard gelatin or soft capsules. It is alsofeasible to prepare the active ingredient of present compounds intomicrocapsule, into suspensions by suspending into aquose medium or intohard capsules or injection formulation.

For example, present extracts or compounds may be prepared to beinjections in form, such as solutions, Suspensions, emulsions andfreeze-dry powder injections. This kind of preparation can contain wateror no water, may contain one and/or multiple pharmacologicallyacceptable carriers, dilutes, adhesives, lubricants, preservatives,surfactants or dispersers. For dilutes, water, ethanol, polyethyleneglycol, 1,3-propylene glycol, ethoxylated prisorine, polyoxylatedprisorine and polyoxyethylene sorbitan alphate are included. Inaddition, it is feasible to add appropriate amounts of sodium chloride,glucose or glycerol into the injections to prepare the isotonicinjections. Furthermore the conventional solubilization boosters,buffering agents and pH adjusting agents may be added. Thesesupplemental materials are commonly used in this field.

Besides, if necessary, the colorants, preservatives, spices, tastemodifiers, sweeteners and other substances may be also included in thepresent pharmaceutical formulations.

For the purposes of achieving the medication goals and improving thetherapeutic effects, present drugs or composition may be administered byany well-known dosing method.

The dosages of the present compounds or composition are determined bymany factors, such as the severity of disease courses for the AIDSpatients, sex, age, body weight, disposition, individual response,dosing routes, dosing frequency and treatment goals. As a result, thetherapeutic dose range of this invention is large in change. Generallyspeaking, the practical doses of drug ingredients of this invention inthe traditional medicine are well known among the professionals in thisfield. It is possible to make adequate adjustments to the actual drugquantity of the final preparation of present compounds or composition soas to achieve the effective therapeutic levels and accomplish thepreventive or therapeutic goals of this invention. The proper daily doserange of the medicinal herbs composition is in amount of 0.03-0.50 mg/Kgbody weight. The above dose may be administered in 2, 3 or 4 times perday. The administration is subject to the clinical experiences ofphysicians and influenced by the dosing plans through other therapeuticapproaches.

It is also possible to prepare those compounds that are more active,fewer amounts found in present invention by semi-synthesis method toprovide sufficient amount of said compounds for pharmaceutical use.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is analysis figure showing the target of Rb3 on inhibiting HIV invitro

FIG. 2 A, B are figures showing the binding of JHR, Rbs to protein gp41.

FIG. 3 is figure showing the binding of Rb3 to protein gp120.

FIG. 4 is figure showing no action of JHR on CD4 receptor, wherein A isfor control, B is for administration.

FIG. 5 is figure showing the effects of the JHR upon the CXCR4 and CCR5co-receptors.

FIG. 6 is analysis figure showing the target of Rb3 on inhibiting HIV invitro.

DETAILED DESCRIPTION OF THE INVENTION

Two aspects were considered by the inventors when carried out researchon the anti-AIDs drugs, one was modern medicine which puts emphasis onthe importance of anti-virus in the treating of AIDs, another was theeffect of drugs on immune function. It is desired, in combination withthe traditional medicinal theory, to provide drugs that have anti-virusactivity and enhanced immune function, and so to achieve the aim ofadjusting the energy flows, nurturing the blood, quieting the spirits,sharpening the wisdom, generating the saliva, relieving the cough,replenishing the nutrients and strengthening the body, and increasingCD4 cells. As a result, the inventors found the compounds of formula I

wherein, R₁ is -Glc²-Glc R₂ is selected from the group of -Glc⁶-Glc,-Glc⁶-Ara(p), -Glc⁶-Xyl and -Glc⁶-Ara(f); and R₃ is H. Preferably, R₁ is-Glc²-Glc, R₂ is -Glc⁶-Xyl, and R₃ is 1H.

Present compounds are used for treating AIDs.

Compounds of formula I are well known, and may be extracted orsynthesized according to the method reported in prior art.

Specifically, the prefer compounds of present invention include Rb1,Rb2, Rb3, Rc and the like, wherein Rb2 and Rb3 are known isomers havingthe same structure.

Said compounds of formula I were extracted first from ginseng. It isfound however that many kinds of plants comprise this kind of compounds,said plants include but do not be limited to: Panax ginseng C. A. Mey,Radix panacis quinquefolii, Gymostemma pentaphyllum (thumb) makino,Panax notoginseng (burk.) F H Chen, Panax japonicus C. A. Meyer andPanax japonicus var: major (burk) Wu et Feng, as well as any other onethat comprises the compounds of formula I. And the compounds of formulaI are not only exit in the root of above plants, but also in the stemand leaf in a sufficient amount.

Present composition comprises more than one compounds of formula I:

wherein, R₁ is -Glc²-Glc, R₂ is selected from the group of -Glc⁶-Glc,-Glc⁶-Ara(p), -Glc⁶-Xyl and -Glc⁶-Ara(f); and R₃ is 1.

The prefer ratio of the components in present composition are: Rb1:15-20wt %, Rb2:15-20 wt %, Rb3:30-90 wt %, Rc:30-90 wt %.

Therefore, skilled person in the art will understand that since thecompounds with formula I have the activity of anti-AIDs, any plantcomprising the compound can also be used to treat AIDs, and as a result,herb composition, mixture or grindings that comprises said compound offormula I are all covered by present protection scope.

The following examples are to explain that present compounds have theactivity of anti-AIDs virus, but not to limit its scope in any manner.

EXAMPLE 1 In Vitro Experiment

Three types of cells (such as MT4, Hela-CD₄, PBMC) were infectedrespectively with HIV-1 virus for observation of the inhibiting effectsof ginseng composition (JHR) prepared in Example 1 upon the HIV-1replication.

(I) MT4 Cells Viral Strains: NL4

Method: A ginseng composition was prepared into a concentration of 1mg/ml as drug solution and diluted into different concentrations forlater uses during the experiment. Based on the analysis, the ginsengcomposition comprises 15 wt % Rb1, 33 wt % Rc, 17 wt % Rb2 and 35 wt %Rb3.

The experiment was carried out on a 96-well culture plate, 100 μl drugsolution was added into each well and making each concentration's drugsolution at least duplicate.

In the tube 5×10⁶ MT4 cells in were infected with HIV-1 (1×10⁴ TCID₅₀ in1 ml medium), then cultured in the incubator with 5% CO₂ at 37° C. for 2hrs, the HIV-1 infected cells were centrifuged, the supernatant wasdiscarded and washed once with RPMI₁₆₄₀ to remove the free virus, 10 mlcomplete medium was added to the HIV-1 infected cells to make cellssuspension (HIV-cell), 100 μl this cell suspension was added into eachwell which has contained drug solution, then the 96-well culture platewas cultured at 37° C. within a CO₂ incubator. On the third day, 100 μLsupernatant was sucked out from each well replaced by 100 μL of mediumwith the same concentrations of drug as one in the wells. 100 μL ofculture medium was added as a control group. On the sixth day, thesupernatant was taken from each well. The Microelisa method and reagentswere adopted to measure the amount of P₂₄ antigen. For each experiment,the virus control (VC), cell control and AZT positive drug control wereused. Based upon the amount of P₂₄ antigen (P₂₄-Ag), the inhibition rate(IR) was calculated according to the following formula:

${I\; R} = {\frac{{V\; C\mspace{14mu} {well}\mspace{14mu} P_{24}\text{-}{Ag}} - {{Drug}\mspace{14mu} {group}\mspace{14mu} P_{24}\text{-}{Ag}}}{V\; C\mspace{14mu} {well}\mspace{14mu} P_{24}\text{-}{Ag}} \times 100}$

Different IRs were obtained for drug solutions of differentconcentrations. After statistic processing, IC₅₀ was obtained.

To MT4 cells, IC₅₀ of ginseng composition JHR is 105.2 μg/ml

(2) Hela-CD4 Cells

Virus: Single-life-cycle reporter HIV was acquired by transfection withHIV plasmids.

Method: Hela-CD₄-LTR-gal cell was inoculated into a 24-well plate at0.4×10⁵/well and cultured for 24 hours and let the cells absorb andadhere to the wall. On the second day, the supernatants were sucked awayfrom the wells and 100 μl drug (drug control) or drug (drug solutionswith different concentrations) and HIV-1 or culture medium (Mock) wereadded. After 2 hours, 200 μl of identical drug solutions or culturemedium was added into each well for culturing at 37° C. in a CO₂incubator for 48 hours and detecting by the following method.

Fixation: The supernatants were sucked away from each well and addingthe fixative solution (1 ml), and then by staining withK₄[Fe(CN)₆].3H₂O, K₃[Fe(CN)₆] and X-gel.

Counting: For the blue cell counts (BCC) within each well, the followingformula is used to calculate the IR and then IC₅₀.

${I\; R} = {\frac{{{Mock}\mspace{14mu} {well}\mspace{14mu} B\; C\; C} - {{Drug}\mspace{14mu} {well}\mspace{14mu} B\; C\; C}}{{Mock}\mspace{14mu} {well}\mspace{14mu} B\; C\; C} \times 100}$

TABLE 1 In vitro: inhibiting the replication of HIV-1 in the cell(HeLa-CD4 cell) (MAGI test) Dose (mg/ml) Inhibition rate (%) 1. 0.5020100 2. 0.1255 98.8 3. 0.0313 26.2 4. 0.0078 0

Result: To Hela-CD4 cell strain IC₅₀ of ginseng composition JHR is 44.3μg/ml.

(3) PBMC Cells

-   -   Virus: NL4-3    -   Method: The freshly harvested PBMC (human peripheral blood newly        isolated lymphocytes) was collected, following by counting and        centrifuging at 1200 rpm, discarding the supernatant and        preparing the medium with 3×10⁶ cell/ml. The culture medium was        pre-treated with IL2 (1 μl 1000×IL2 for every ml culture medium)        at 37° C. overnight. For the experiment, 5×10⁶ was counted as        one infection unit. The duplicate series of JHR at the same        concentration of 0.4 mg/ml, virus control and cell control were        set up. On a 24-well plate, each well contained 5×10⁶ cells in        0.5 ml drug solution or culture medium. NL4-3 virus (HIV-1) with        the viral load of 4×10⁴ IU in each well was mixed and        transferred into a 12-well plate. Then 1.5-ml of the identical        drug solution or culture medium were added and incubated at        37° C. The supernatant was taken every 3-4 days in 100 μl        Fetched for each well and stored at −80° C. RT was measured and        the drug group was compared with virus control group to        calculate the inhibition rate.

Result: Ginseng composition has outstanding inhibition activity to theHIV-1 of PBMC cell in vitro test shown in Table 2.

TABLE 2 Days after administration inhibition rate (%) 3 days 73.59 6days 100 9 days 100 12 days  100

EXAMPLE 2

The same method as that in example 1 is applied, except that the drugadded into the wells is Ginsenoside monomer, and the concentrationthereof is listed in table 3.

(I). Analysis and Comparison of Anti-AIDs Effect of GinsenosideMonomer-Hela-CD4 Cell Strain

TABLE 3 Inhibition Rate Inhibition Rate Compound (0.2 mg/ml) (0.4 mg/ml)Rb1 41.88% Rb2 42.93% Rb3 70.68% 97.9% Rc 60.73 Rd 9.42%

It can be seen from the above table, among five Ginsenoside extracts,Rb3 has the best inhibition rate of anti-AIDs.

(II) Action Targets of Rb3

Object: It is to observe which phase(s) of viral life cycle are targetedby the said ginseng composition, including viral entry into cells,reverse transcriptase, integrase, transcription and proteases. The viral“single life cycle” model was employed to study the target of drugactions.

(1) MAGI method: The recombinant virus has the LTR of HIV. The reportergene of β-galactosidase was expressed to form one kind of vital “singlelife cycle” model. As mentioned as above, this model employsK₄[Fe(CN)₆], K₃[Fe(CN)₆] and x-gel to stain the Hela-CD₄ cell. The bluecells under the microscope denote the presence of viral replication.

(2) Luciferase Method

The recombinant and transfected VSVG virus and the cell line of H9strain were adopted. The examination method was used to detect theactivity of Luciferase by illumination. A heavier viral load denoted ahigher enzymatic activity.

Experiment method: After viral infections, the cells were divided intodifferent groups. They were also dosed respectively at 0, 6, 12, 18, 24and 36 hours after infections. 48 hours after infection, the method ofMAGI or Luciferase was adopted.

The above two testing methods were both viral models of “single lifecycle”. Their major advantages showed that different infection timesdenoted different phases of viral replications. At 2-6 Hours, the viruswas entering the cell. At 10-14 Hours, it was the phase of reversetranscription. After 20 Hours, it was the phase of recombination andtranscription. As a result, dosing at different time points acted uponspecific target points. This invention experiment had analyzed differenttarget points of JHR and Rb3.

TABLE 4 Analysis on the viral life cycle stage(s) targeted by drug ofRb3 disclosed in present invention Days after dosing (post-infection) 2hours 10 hours 29 hours Inhibition rate (%) 83.98 0 0

The result is shown in FIGS. 1 and 6, and it can be seen from thefigures that the best inhibition effect was obtained when the drugs wasadministrated after 2 hours of the infection, with the target at thattime is to block the viral to entry into the cells.

Macrophage-tropic lymphocytes were used to show the action on CCR5co-receptor, and T lymphotropic lymphocytes are used to show the actionon CXCR4 co-receptor.

(III) The use of BIACORE in Measuring the Binding of JHR, Rb3 to gp41,gp120 Protein1. The Binding of Rb3 to gp41 Protein

The genetically recombined gp41, gp120 were placed on the chip ofBIACORE analyzer, respectively, after adding a certain concentration ofRb3 in sufficient amount, the instrument could detect whether or notthere was a binding. The result is shown in FIG. 2

2. The Binding of Rb3 to gp120 Protein, the Result is Shown in FIG. 3.

From the result, it can be seen that both JHR and Rb3 can bind to gp41and gp120 protein, with the conjugation to gp41 protein stronger.

(IV) The Action of JHR on CXCR4 Receptor by T Lymphotropic Lymphocytesand on CCR5 Co-Receptor by Macrophage-Tropic Lymphocytes

The test was carried out by using the same method as MAGI test above.(The result is shown in FIG. 5), the explanation to the result is listedin the following Table 5:

TABLE 5 The study on the co-receptor and mechanism of entry thereof(MAGI test) T-cell lymphotropic virus strain (NL4). Hela-CD4 cellMacrophage-tropic virus strain (Yu2). 293 T cell Drug Virus Cell strainResults Drug NL4 Hela-CD4 Inhibition No inhibition Inhibition Drug Yu2293T cell No inhibition Inhibition Inhibition Expla- Acting on Acting onActing on nation CXCR4 CCR5 CD4 or co-receptor co-receptor othermechanism Other mechanism: acting on, other than receptor, cellmembrane, coat of virus, releasing of RNA, etc.

(V) Action of JHR on CD4 Receptor

The method of Flow Cytometry was used for measurement. Method was givenas follows: SupT1 cell with drug was co-incubated at 37° C. for 2 hoursand washed with PBS+2% FCS. In a 4° C. ice bath, CD₄PE was added and sitfor 30 minutes. After further washing and centrifuging, CD₄ monoclonalAb was added to, followed by incubating in ice bath for 30 minutes;again after washed and centrifuged and sit in ice bath. The cells weresuspended in 50 μl secondary Ab anti-mice-FITC for 20 minutes, followedby washing once and were suspended in 300-500 ul PBS/2% CS+PI. FACStesting was performed. The testing results were shown in FIGS. 4A, B.

Results showed that JHR had no effects upon CD4 receptor.

EXAMPLE 3 Study on Combination Medication

Object: It is to observe if there is any synergistic effect between JHRand AZT.

Method: For the experiment, the MAGI test method was adopted (the sameas above).

(A) Single-agent medication: five doses of AZT were used in amount offrom 1 μM to 3.9 nM, and were designated AZT1-5, five doses of JHR weredesignated in amount of 400 ug/ml to 1.56 ug/ml, and IC₅₀ were obtainedrespectively.

(B) Combined Application

Half dosage of AZT was combined with half dosage of JHR-1 as one sample.AZT1 was combined respectively with any of JHR1-JHR5; Each of AZT2,AZT3, AZT4, AZT5 was respectively combined with any of ZN1-ZN5. So atotal of 25 concentration combinations could be used. Each concentrationwas set in duplicate wells (2 wells). Additional group was taken as celland virus controls.

(C) Comparing each drug combination with the virus group to have theinhibition rate. The inhibition rate of each drug combination wascompared with the sole AZT IC50 to obtain the difference of functionseach other.

Result: see table 6

TABLE 6 Drug ED50 AZT/A + J JHR 21.5 μg/ml AZT 46 nM AZT-JHR1 5.8 nM7.93 AZT-JHR2 10 nM 4.60 AZT-JHR3 16.7 nM 2.75 AZT-JHR3 33.5 nM 1.37AZT-JHR-5 40.6 nM 1.13

As shown in Table 6, the IC50 of AZT alone is 46 nM/ml, after combiningwith the first dose of JHR (the composition is the same with that inexample 1), IC50 of AZT is only 5.8 nM/ml, that is, ⅛ dose will obtainthe same effect, showing that there is synergistic effect between JHRand AZT.

EXAMPLE 4 Effects on the HIV Strains Resistant to Drugs

HIV-1 was the strain resistant to protease inhibitor with virulence at5.7×10⁴ IU/ml. The Hela-CD4 cell was adopted in MAGI test in order toobserve the effects of JHR and see if there was any cross-reactivity.

Results indicated that dose of JHR was 0.4 mg/ml and the inhibitionrates of virus for 5 μl or 8 μl were both as high as 100%. Thesedemonstrated that JHR could effect upon the HIV strains resistant toprotease inhibitors. The results refer to Table 5.

TABLE 5 Inhibition rate of JHR to HIV strains resistant to proteaseinhibitors Virus load Drug Inhibition rate % PRIV 5 μl JHR0.4 mg/ml 1008 μl JHR0.4 mg/ml 100 Note: PRIV is the viral strain resistant toprotease inhibitors.

The result shows that JHR is well effective in inhibiting HIV-1 strainsresistant to protease inhibitors.

EXAMPLE 5 Toxicity Experiment I. Acute Toxicity Experiment

Result: Acute toxicity experiment indicated that no toxicity wasobserved for doses over 20 g/kg in intragastric dosing for rats.

II. Sub-Acute Toxicity Experiment

Result: Sub-acute toxicity experiment indicated that after continuousintragastric dosing for 6 months, rats grew normally in the large,medium and small dose groups, ALT, BUN, RBC, WBC with DC were all normaland no abnormality was observed on pathological slides for organs suchas heart, liver, kidney, spleen, lung, pancreas, brain, testis andovary.

INDUSTRIAL APPLICABILITY

Ginsenosides of present invention can be used to prepare drugs fortreating AIDs.

1. Use of compounds of formula I in the preparation of drugs fortreating AIDs,

wherein, R₁ is -Glc²-Glc, R₂ is selected from the group of -Glc⁶-Glc,-Glc⁶ Ara(p), Glc⁶-Xyl and -Glc⁶-Ara(f); and R₃ is 1-1H.
 2. The useaccording to claim 1, characterized in that R₁ is -Glc²-Glc, R₂ is-Glc⁶-Xyl, and R₃ is H.
 3. The use according to claim 2, characterizedin that dosages of said compounds are in amount of 0.03-0.50 mg/kg bodyweight.
 4. The use according to any of claims 1-4, characterized in thatsaid compounds are combined with commercialized anti-HIVs drugs.
 5. Theuse according to claim 4, characterized in that said anti-HIVs drugs areselected from the group of AZT, DDC, DDI, Saquinavir, Ritonavir,Indinavir sulfate and Nefinavir or combination thereof.
 6. The useaccording to claim 4, characterized in that said HIVs are thoseresistant to drugs repeatedly used to anti-HIV.
 7. Use of compositioncomprising compounds of formula I as active ingredient in thepreparation of drugs for treating AIDs,

wherein R₁ is -Glc²-Glc, R₂ is selected from the group of Glc⁶-Glc,-Glc⁶-Ara(p), -Glc⁶-Xyl and -Glc⁶-Ara(f); and R₃ is H.
 8. The useaccording to claim 7, characterized in that the composition is combinedwith commercialized anti-HIV drugs.
 9. The use according to claim 8,characterized in that said anti-HIVs drugs are selected from the groupof AZT, DDC, DPI, Saquinavir, Ritonavir, Indinavir sulfate and Nefinaviror combination thereof.
 10. The use according to claim 9, characterizedin that said HIVs are those resistant to drugs repeatedly used toanti-HIV.
 11. Use of root, stem or leaf extracts and/or dry powder ofplants selected from the group of Panax ginseng C. A. Mey, Radix panacisquinquefolii, Gymostemma pentaphyllum (thumb) makino, Panax notoginseng(burk) F. H. Chen, Panax japonicus C. A. Meyer and Panax japonicus var:major (burk.) Wu et Feng in the preparation of drugs for treating AIDs,characterized in that the extracts comprise compounds of formula I

wherein, R₁ is -Glc²-Glc, R₂ is selected from the group of -Glc⁶-Glc,-Glc⁶-Ara(p), -Glc⁶-Xyl and -Glc⁶-Ara(f); and R₃ is H.
 12. The useaccording to claim 11, characterized in that said extracts are combinedwith commercialized anti-HIVs drugs.
 13. The use according to claim 11,characterized in that said anti-HIVs drugs are selected from the groupof AZT, DDC, DPI, Saquinavir, Ritonavir, Indinavir sulfate and Nefinaviror combination thereof.
 14. The use according to claim 13, characterizedin that said HIVs are those resistant to drugs repeatedly used toanti-HIV.